Mixed-species communities of microbes are examples of genome-genome interactions. Such communities in the human oral cavity form dental plaque. We are expanding our previous studies that demonstrated initial dental plaque is composed of several kinds of interacting cells. During this reporting period, novel plasmids for use in Fusobacterium nucleatum, an oral microbe that interacts with a wide variety of other human oral bacteria including initial colonizers such as oral streptococci and actinomyces, have been developed. We hypothesize that dental plaque communities initiate through intimate interactions between cells (genomes) of different species and not by clonal growth of genetically identical cells. The human oral cavity provides easy access to natural biofilms on a retrievable enamel chip, which is an excellent model to study genome-genome interactions. To complement our studies of initial oral microbial communities formed on the retrievable enamel chip, we tested the role of cell-cell interactions in forming multispecies communities in a saliva-conditioned in vitro flowcell. We had shown previously that a pair of streptococcal and actinomyces species exhibited mutualism when grown together on saliva as the sole nutrient source; individually neither species could grow on saliva. In this reporting period, we extended this aspect of the study to examine a community of four oral bacterial species for additional potential for mutualism when grown in a flowcell with saliva as the sole source of nutrition. All four species interacted and formed mixed-species communities. Again two species were found to mutually benefit by being inoculated together into the flowcell, suggesting that oral bacteria communicate through close contact and metabolic products. We have continued our studies on early colonizing streptococci. In this reporting period, we examined the effects of antimicrobial agents on a streptococcal biofilm grown in a saliva-conditioned flowcell. The flowcell coupled with confocal laser microscopy enabled examination of growing oral biofilms in situ without disruption of the microbial community. Biofilms composed of oral streptococci were grown in the flowcell and treated with several commercially available antimicrobial mouthrinses. The results of this study revealed varying abilities of the antimicrobial agents to cause cellular damage on the growing biofilm in situ, and this study demonstrated the usefulness of the flowcell in the rapid assessment of antimicrobial efficacy. We are continuing our investigations of cell adherence molecules, cell signaling molecules, gene expression within human oral biofilms, and of the relevance of these events to bacterial community architecture. Our hypothesis is that signaling molecules involved in cellular communication impact the spatiotemporal development and establishment of dental plaque as well as colonization of the host. One pair of early colonizing species includes streptococci which ferment complex carbohydrates and sugars to lactic acid, which is the preferred substrate for growth by veillonellae. Initial results obtained during this reporting period suggest that veillonellae send a signal to streptococci that induces the expression of a gene encoding an amylase. Microbial diversity and accessibility make dental plaque communities very attractive for the study of genome-genome interactions, which might occur at multiple levels including physical, metabolic, and genetic. We emphasize here the importance of determining first the physical interactions occurring in vivo between cells of different species and genera in order to design the correct in vitro studies to determine potential metabolic exchange between species. Our long-range goal is to understand the molecular mechanisms of cellular communication and their relationship to the spatiotemporal development and establishment of dental plaque andd colonization of the host epithelial cells.